Lecture 3

Question 1

Examples of when you need recombinant proteins and specific examples of these.

Answer 1

Badly worded question get over it
therapeutic proteins - insulin, interferon Beta, Factor VIII
enzymes for biotech - lipases, proteases, DNAse I, Taq and restiriction enzyme
and protein you wish to study

Question 2

How did insulin used to be got?

And why was this bad

Answer 2

Extracted from animals
needed lots of animals just to treat one person
contamination risks

Question 3

Name the longer peptide insulin is derived from

Answer 3

preproinsulin

Question 4

How is insulin made now?

Answer 4

Vector carrying LacZ promoter, lacz gene, and gene for peptide A icloned into E.coli expression vector.
Meanwhile vector carrying the same but B peptide instead of A is cloned and synthesised in another E.coli vector.
These proteins, when synthesised by Ecoli are fused to Beta - galactosidase (just like notmal fusion protein)
Each purified separately and fusion region cleaved off
peptides then mixed together and they become attached together

Question 5

By what re the 2 peptide chains linked together

Answer 5

Disulphide bonds

Question 6

Why is a fusion protein added and what is that fusion method officially called?

Answer 6

N terminal fusion

Question 7

State the generic steps from a cloned gene to a functional protein

Answer 7

DNA -transcription (RNA pol) -mRNA - translation (ribosome) - protein -folding, localisation and purification (must purify active form) - functional protein

Question 8

What regions on the genome must an expression vector have?

Answer 8

Promoter region (TATA) - ribosome binding site (Shine D) - start codon (AUG) - what you want to express - stop codon (UAAU) - transcriptional terminator
Bracketed parts specifically for prokaryotes

Question 9

What properties must a good expression vector have

Answer 9

• High plasmid copy number
• Strong promoter - where RNA pol binds to initiate ‘scription. Often use tac (hybrid of lac and trp).
• Transcription terminator - cos have other genes you don’t want to express and don’t want to waste energy making more mRNA. Is a hairpin loop which kicks RNA pol off DNA.
• Efficient translation initiation - SD, start and stop codons
• Stable transcript - most bacterial mRNA short half life (so they can respond fast to stress). This’ll be covered in another question before you get carried away
• codon usage - some Ecoli fussy about which codon which aa they use. To get around this remove unwanted codons by SMD (time consuming) or add extra tRNA

Question 10

How is a stable transcript achieved?

Answer 10

addition of hairpin forming 5’ UTR which reduces degradation by RNAasE and prevents polyadenylation which would normally promote rapid degradation by RNaseII

Question 11

What is the importance of a regulated promoter?

Answer 11

prevent expression when not wanted, i.e. in the wrong growth phase
To keep a minimal level of basal expression - don’t want any ideally.

Question 12

What are the 2 main mechanisms of regulating expression?

Answer 12

Lac repressor - LacI

pET vector system

Question 13

which is better?

Answer 13

pET

Question 14

Describe using the LacI repressor

Answer 14

LacI binds to promotor, prevents RNA pol from binding.
Lactose is the physiological inducer - if it is present LacI falls off
‘Scription and ‘Lation can occur

Question 15

What is the main disadvantage of the LacI repression system?

Answer 15

Ecoli eats lactose so even if it’s present LacI might not drop off because lactose is gobbled up too soon.

Question 16

What is a gratuitous inducer, and name the one for the LacI repression system

Answer 16

An inducer structurally and molecularly similar to another and can induce in it’s absence.
Isopropyl-beta-D-thiogalactopyranoside (IPTG)

Question 17

Describe the pET vector system for expression regulation

Answer 17

T7 polymerase gene included in Ecoli chromosome -> codes for special RNA pol. Controlled by Lac promoter.
Insert plasmid expression vector containing GofI and T7 late promotor.
T7 gene in chromosome transcribed and t7 RNA pol formed. Attaches to the T7 late promotor on foreign plasmid, GofI is expressed (downstream from the promotor)